1. Field of the Invention
This invention relates to rationally designed mixed mode resins which are useful in recovering a target compound from an aqueous solution and methods for use of such resins. The resins described herein have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound from the resin and are specifically designed to bind the target compound from an aqueous solution at both a low and high ionic strength.
2. References
The following references are referred to in the specification as numbers in []:
1 Ochoa, J. L., "Hydrophobic (interaction) chromatography", Biochimie, 60:1-15 (1978). PA0 2 Yon, R. J., et al., "Protein Chromatography on Adsorbents with Hydrophobic and Ionic Groups", Biochem J., 151:281-290 (1975). PA0 3 Yon, R. J., "Chromatography of Lipophilic Proteins on Adsorbents Containing Mixed Hydrophobic and Ionic Groups", Biochem J., 126:765-767 (1972). PA0 4 Hofstee, B. H. J., "Hydrophobic Affinity Chromatography of Proteins", Anal. Biochem., 52:430-448 (1973). PA0 5 Hofstee, B. H. J., "Protein Binding by Agarose Carrying Hydrophobic Groups in Conjunction with Charges", Biochem. Biophys. Res. Commun., 50:751-757 (1973). PA0 6 Bischoff, et al., "Nucleic Acid Resolution by Mixed-Mode Chromatography", J. Chromatogr., 296:329-337 (1984). PA0 7 Kasche, V., et al., "Rapid Protein Purification Using Phenylbutylamine-Eupergit: a novel method for large-scale procedures", J. Chromatogr., 510:149-154 (1990). PA0 8 Sasaki, I. et al., "Hydrophobic-Ionic Chromatography", J. Biochem., 86:1537-1548 (1979). PA0 9 Sasaki, I. et al., "Hydrophobic-Ionic Chromatography: Its Application to Microbial and Glucose Oxidase, Hyaluronidase, Cholesterol Oxidase, and Cholesterol Esterase", J. Biochem., 91:1555-1561 (1982). PA0 10 Simons, P. C. et al., "Purification of Glutathione S-Transferases from Human Liver by Glutathione-Affinity Chromatography", Anal. Biochem., 82:334-341 (1977). PA0 11 McLaughlin, "Mixed-Mode Chromatography of Nucleic Acids", Chem. Rev., 89:309-319 (1989) PA0 12 Bischoff, et al., "Mixed-Mode Chromatographic Matrices for the Resolution of Transfer Ribonucleic Acids", J. Chromatogr., 317:251-261 (1984) PA0 13 Champluvier, B., et al., "Dye-Ligand Membranes as Selective Adsorbents for Rapid Purification of Enzymes: A Case Study", Biotechnol. Bioeng., 40:33-40 (1992) PA0 14 Butler, L. G., "Enzyme Immobilization by Adsorption on Hydrophobic Derivatives of Cellulose and Other Hydrophilic Materials", Arch. Biochem. Biophys., 171:645-650 (1975). PA0 15 Caldwell, K. D., et al., "Utilization of Hydrophobic Interaction for the Formation of an Enzyme Reactor Bed", Biotechnol. Bioeng., 17:613-616 (1975). PA0 16 Cashion, P., et al., "Enzyme Immobilization on Tritylagarose", Biotech. Bioeng., 24:403-423 (1982). PA0 17 Voutsinas, P. L., et al., "Coagulation of Skim Milk with Proteases Immobilized on Hydrophobic Carriers", Dairy Sci., 66:694-703 (1983). PA0 18 Hutchinson, D. W., "The Preparation and Properties of Immobilized Dipeptidyl-aminopeptidase I (cathepsin C)", Biochim. Biophys. Acta, 916:1-4 (1987). PA0 19 Asenjo, J. A., et al., "Rational Design of Purification Processes for Recombinant Proteins", Ann. N.Y. Acad. Sci., 646:334-356 (1991) PA0 20 Ruann, R. C., et al., "Dual-Functional Affinity Protein Purification", Biotechnol. Prog., 4:107-112 (1988) PA0 21 Teichberg, V. I., "Affinity-Repulsion Chromatography", J. Chromatogr., 510:49-57 (1990) PA0 22 Johansson, G., et al., "Affinity Partition Between Aqueous Phases--A Tool for Large-Scale Purification of Enzymes", J. Biotechnol., 11:135-142 (1989) PA0 23 Ortin, A., et al., "Large Scale Extraction of a .alpha.-Lactalbumin and .beta.-Lactoglobulin from Bovine Whey by Precipitation with Polyethylene Glycol and Partitioning in Aqueous Two-Phase Systems", Prep. Biochem., 22:53-66 (1992) PA0 24 Heath, C. A. et al., "Synthetic Membranes in Biotechnology: Realities and Possibilities", Adv. Biochem. Eng./Biotechnol., 47:45-88 (1992) PA0 25 Luong, J. H. T., et al., "Synthesis and Characterization of a Water-Soluble Affinity Polymer for Trypsin Purification", Biotechnol. Bioeng., 31:439-446 (1988)
The disclosure of each of the above-referenced publications is hereby incorporated by reference in its entirety to the same extent as if each and every reference were individually incorporated by reference herein.
3. State of the Art
In recent years, several techniques have been developed and/or optimized to effect separation and purification of target compounds from an aqueous mixture. Separation and purification techniques heretofore employed with such compounds include, by way of example, ion-exchange chromatography, hydrophobic interaction chromatography (HIC) [1], affinity chromatography, and the like. The multiplicity of such chromatographic techniques reflect the difficulty in effecting separation and/or purification of the target compounds while minimizing the complexity of the separation/purification procedure and each of the techniques recited above suffer from one or more drawbacks limiting their broad use on an industrial scale.
Mixed mode chromatographic resins have also been employed in the art wherein such resins effect binding of a target compound under hydrophobic conditions and effect desorption of the target compound from the resin under electrostatic (ionic) or hydrophilic conditions. Examples of such mixed mode resins are found in Kasche, et al. [7], Bischoff, et al. [6], McLaughlin [11] and Bischoff, et al. [12].
One problem typical of mixed mode chromatographic resins of the prior art is that binding efficiencies of less hydrophobic target compounds to the resin is not very high unless a high salt concentration is employed in the target compound solution. For example, in Kasche, et al. [7], protein binding to the resin on a preparative level was effected using a 1M NaCl solution. Chromatographic techniques involving the addition of salt to an aqueous solution containing the target compound require the use of large quantities of reagents to effect recovery on an industrial scale and may necessitate substantial processing. Accordingly, chromatographic resins requiring the use of high salt concentrations are not the most efficient and cost effective methods for recovering and/or purifying industrial quantities of such compounds.
This invention is directed, in part, to the discovery that the use of a high ligand density on a mixed mode chromatographic resin coupled with the use of a specific ionizable ligand comprising an ionizable functionality and a spacer arm which covalently links the ligand to the solid support matrix of the resin can provide for sufficient hydrophobic character in the resin such that the target compound binds to the resin at high and low ionic strength. This invention is further directed, in part, to the discovery that target compound desorption from the resin can be achieved by hydrophilic or electrostatic (ionic) interactions by merely altering the pH of the desorbing solution such as to increase the amount of charge on the resin via the ionizable functionality. This latter feature provides a significant advantage over high density HIC resins by providing a facile means to provide product recovery whereas binding to such HIC resins can often be irreversible.
In this invention, the ionizable ligand is rationally selected relative to the target compound thereby permitting the efficient large scale recovery and/or purification of a target compound from an aqueous medium including a fermentation broth. The resins employed herein are specifically characterized as comprising a solid support matrix and selected ionizable ligands or mixtures of selected ionizable ligands covalently attached to the solid support matrix at a density greater than the smaller of either at least 150 .mu.mol per milliliter of resin or 1 mmol per gram dry weight of resin. The ionizable ligands comprise a spacer arm and at least one ionizable functionality which functionality is bound to the matrix through the spacer arm. The ionizable ligand employed on the resin is selected relative to the solid support matrix and the target compound such that the hydrophobic character of the resin is sufficient to bind at least 50% of the target compound in an aqueous medium at high and low ionic strength at a first pH and is further selected relative to the solid support matrix and the target compound such that the hydrophilic and/or electrostatic (ionic) character of the resin at a second pH is sufficient to desorb the bound compound at said second pH. Additionally, the ionizable functionality is partially electrostatically charged at the pH of binding of the compound to the resin and is either further charged or charged at a different polarity the pH of desorption of the compound from the resin. Such partially charged resins may provide a secondary basis for enhancing or weakening the binding of the target compound to the resin.
In a preferred embodiment, the electrostatic charge on the resin at the pH where the target compound is desorbed from the resin is of the same polarity as the net electrostatic charge on the target compound at the desorption pH. In this embodiment, desorption is achieved by charge-charge repulsions which offset the hydrophobic binding character of the resin. In another embodiment, the electrostatic charge on the resin at the pH where the target compound is desorbed from the resin is of opposite polarity from that of the target compound. In either embodiment, desorption can be facilitated by use of desorbing solution (e.g., eluants) of high ionic strength or by use of a polarity reducing agent, such as ethylene or propylene glycol.
The resins of this invention are contrasted with mixed-mode resins heretofore described by the character that the resin comprises a ligand density greater than the smaller of either at least 150 .mu.mols per milliliter of resin or 1 mmol per gram dry weight of resin and further by the character that the ligand contain a spacer arm which covalently links an ionizable functionality to the matrix. Specifically, in the literature, ionizable groups have been deliberately introduced [2,3] to weaken strong binding to long alkyl chain (hydrophobic) Sepharose resins and allow more favorable desorption conditions. The ligand density on these matrices has been calculated to be about 15-21 .mu.mol per milliliter.
A polystyrene carboxyl resin (Amberlite) has also been used for protein binding by hydrophobic interactions [8,9] and desorption by ionic interaction. However, while a high density of carboxyl groups is employed, there is no spacer arm linking the carboxyl groups to the solid support matrix.
Also disclosed are resins which are positively charged with isourea groups [4,5] having a density believed to be about 20 .mu.mol per milliliter or less. These resins are weakly hydrophobic and typically require electrostatic and hydrophobic interactions for binding of the target compound to the resin.
Binding of target compounds at high and low ionic strength to the resins of this invention which resins contain ionizable functionality is in contrast to the resins of the prior art having ionizable functionality which typically require adjustments in the ionic strength of the solution either prior to binding of the target compound or to effect desorption of the target compound from the resin. Such adjustments are not consistent with an efficient industrial scale process to effect target compound recovery and/or purification.